The development of eukaryotic viral vectors has generally focused on delivery of one or more heterologous genes to target cells, particularly for gene therapy applications. A wide variety of viruses have been studied as potential eukaryotic viral vectors, e.g., adenovirus, herpes simplex virus, vaccinia virus, adeno-associated virus, and retrovirus (see, e.g., Berkner, Biotechniques 6: 616-629 (1988); Fisher et al., Human Gene Therapy 7: 2079-2087 (1996); and Fassati et al., Retroviral Vectors, Molecular and Cell Biology of Human Gene Therapeutics pp. 1-19 (Dickson ed., 1995)). Each of the vector systems has focused on a strategy that involves: (1) construction of a replication deficient viral vector by removing essential viral genes; (2) replacing these viral genes with a heterologous gene; and (3) transfecting the viral vector into a packaging cell line that complements the deleted viral genes, producing replication deficient viral particles.
Of all the viral vector systems studied to date, retroviral based systems remain the most popular. Retroviral vectors are useful because the genes they transduce are integrated into the genome of target cells, providing long term, stable expression of the heterologous gene. However, retroviral based vector systems have a number of problems. Most importantly, viral titers of retroviral particles produced from packaging cells are low, on the order of 10.sup.6 -10.sup.7 viral particles/ml. In addition, the retrovirus particle often stimulates an immune response because it is derived from a "non-self" packaging cell line.
Other viral based systems have encountered related difficulties. For example, although packaged adenovirus generally has high titers, the viral genome is episomal and transient. In addition, the virus induces an immune response, particularly in the majority of the human population that has been previously infected with this common virus. Similarly, both HSV and vaccinia vectors often provoke an immune response, particularly in those humans that either have been previously exposed to the virus or who have been immunized against the virus.
Adeno-associated viruses (AAV) are naturally occurring replication deficient viruses of the Dependovirus genus, Parvoviridae family. AAV vectors have also been investigated as potential eukaryotic viral vectors. These viruses require co-infection with a helper virus, typically adenovirus or HSV, for production of AAV viral particles from an infected cell. The AAV genome contains two genes, rep, necessary for replication of the viral genome, and cap, a virion protein. These genes are expressed once the AAV particle has infected a target cell.
Recently, Fisher et al., supra, described a vector that is a hybrid between adenovirus and AAV. This hybrid was created by first constructing a replication deficient adenovirus vector that contained an AAV genome, in which the rep and cap genes were replaced by the .beta.-galactosidase gene. Second, this hybrid vector was transfected into 293 host cells, which packaged the adenovirus-AAV hybrid vector into an adenovirus particle. Rep and cap were provided as a separate plasmid tethered to the outside of the adenovirus particle via a polylysine bridge. Finally, this hybrid adenovirus was then used to infect producer host cells. Transient expression of rep by the tethered plasmid allowed rescue and replication of the AAV genome, and production of AAV particles.
However, this system fails to solve the problem of low virus titers and transduction efficiency. The AAV genome lacks the rep and cap genes, expression of which are necessary for efficient AAV replication and integration in the target cell after infection by an AAV virus particle. Instead, a low proportion of the AAV particles contain the rep protein expressed from the tethered plasmid. Thus, few infectious AAV particles are produced from this hybrid system. In addition, rep expression is generally toxic to cells. International patent application PCT/FR95/00233 describes a similar system, with similar defects.
Eukaryotic viral vectors have many applications beyond gene therapy, although these applications have not been extensively investigated. For example, previous studies have suggested that defective retroviral genomes (e.g., defective for gag, pol, or env genes, or the splice signals of the replication competent retroviruses) can be "rescued" from the genomic DNA of cells by infection with replication competent retroviruses, which transcomplement and package the defective retroviral genomes (Wilkinson et al., Endogenous Human Retroviruses, in The Retroviridae (Levy, ed., 1994)). This rescue could provide an important means of diagnosing new disease caused by unknown viruses. However, this rescue is complicated by the difficulty of identifying the defective genomes in the midst of the large number of wild type replication competent retrovirus genomes.
Accordingly, there is a need for improved eukaryotic viral vectors for diagnostic applications and for delivering heterologous genes to cells in vitro, ex vivo, and in vivo. This invention fulfills these and other needs.